Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: A new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was de termined by incubating the reduced forms of the pyridine coenzymes with eit her Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures usin g a HPLC separation of adenine nucleotide derivatives. The effect of the az o-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tes ted. Results showed that, whilst all the three free radical-producing syste ms induced, with different extent, the oxidation of NAD(P)H to NAD(P)(+), o nly Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose( P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 mu M) or H2O2 (concentration range 50-1000 mu M) , were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)(+), ADP-ribo se(P) and nicotinamide formation increased by increasing the amount of hydr oxyl radicals produced in the medium. Under such incubation conditions NAD( P)(+)/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)(+)/AD P-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5 , respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5 s from the beginning of the Fenton reaction. Inhibi tion of ADP-ribose(P), nicotinamide and NAD(P)(+) production of Fe2+-H2O2-t reated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the r eaction mixture. Differently, selective and total inhibition of ADP-ribose( P) and nicotinamide formation was obtained by performing the Fenton reactio n in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Ex periments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hy droxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possi ble mechanism of action of ADP-ribose(P) and nicotinamide generation throug h the interaction between NAD(P)H and hydroxyl radical (which does not invo lve the C-center where "conventional" oxidation occurs) is presented. The i mplication of this phenomenon in the pyridine coenzyme depletion observed i n postischemic tissues is also discussed.